Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences

Discrimination between Demodex folliculorum (Acari: Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences 1 first-author †E-mail:zhaoyae@mail.xjtu.edu.cn †E-mail:zhaoyae@mail.xjtu.edu.cn zhaoyae@mail.xjtu.edu.cn 1 1 1 2 Department of Immunology and Pathogen Biology, Xi’an Jiaotong University College of Medicine, Xi’an 710061, China Department of Immunology and Pathogen Biology, Xi’an Jiaotong University College of Medicine, Xi’an 710061, China Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla 41012, Spain Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla 41012, Spain For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. A new subspecies of Demodex folliculorum has been proposed, but not confirmed. Here, cox1 partial sequences of nine isolates of three Demodex species from two geographical sources (China and Spain) were studied to conduct molecular identification of D. folliculorum . Sequencing showed that the mitochondrial cox1 fragments of five D. folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%. The average sequence divergence was 1.24% among the five Chinese isolates, 0.94% between the two geographical isolate groups (China (5) and Spain (1)), and 2.15% between the two facial tissue sources (facial skin (6) and eyelids (1)). The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125, 2.7 (3/1) among the five Chinese isolates, 0.0094, 3.1 (3/1) between the two geographical isolate groups, and 0.0217, 4.4 (3/1) between the two facial tissue sources. Phylogenetic trees showed that D. folliculorum from the two geographical isolate groups did not form sister clades, while those from different facial tissue sources did. According to the molecular characteristics, it appears that subspecies differentiation might not have occurred and that D. folliculorum isolates from the two geographical sources are of the same population. However, population differentiation might be occurring between isolates from facial skin and eyelids. Demodex folliculorum cox1 partial sequences Divergence Genetic relationship Phylogenetic tree Introduction 1 Demodex is a permanent parasitic mite distributed worldwide, belonging to the mite family Demodicidae of the subclass Acari. The mites infest the hair follicles, sebaceous glands, melbomian glands, ceruminous glands, and internal organs of 11 orders of mammals, including dog, sheep, cat, and pig, etc. For a long time, classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics. Since Berger first identified Demodex in 1841, 140 species or subspecies have been identified (Li, 2009 2009 ). Two or more mite species might simultaneously parasitize the same host. It is generally considered that Demodex is a host-specific obligate parasite. Presently, Demodex cannot be maintained or cultured in vitro so as to parasitize and infect other healthy animal hosts (Zhao et al., 2009a 2009a ; 2011 2011 ). However, there are a few reports of cross-infection between humans and animals (Morsy et al., 1995 1995 ; Wang et al., 1998 1998 ). In 1842, Simon first described the multiforms of Demodex folliculorum , followed by Wilson (in 1844), Hirst (in 1919), and Fuss (in 1933, 1935, and 1937). Akbulatova first described Demodex brevis in 1963, but did not name it, because it was then considered as one of two subspecies of D. folliculorum , including D. folliculorum longus and D. folliculorum brevis (Li, 2009 2009 ). Desch and Nutting ( 1972 1972 ) identified D. folliculorum Simon 1842 and D. brevis Akbulatova 1963, two types of parasitic Demodex mites found in humans, by traditional morphological classification methods. Xie et al. ( 1982 1982 ) proposed a new subspecies, D. folliculorum sinensis , a common human hair follicle mite in China, based on its different morphological characteristics. However, their proposal has neither been confirmed nor applied in later studies. The traditional mite classification based solely on phenotype has obvious limitations as it may be affected by the environment and cannot directly reflect the molecular structural characteristics of genomic DNA. This can cause difficulties and indeterminacy in species classification and lineage. The development of molecular biology techniques has made studies of molecular markers and Demodex species identification at the molecular level feasible. However, our database searches found only a few reports of effective molecular identification of Demodex (Zhao and Cheng, 2009 2009 ; Toops et al., 2010 2010 ; Ravera et al., 2011 2011 ; de Rojas et al., 2012a 2012a ; 2012b 2012b ; Zhao and Wu, 2012a 2012a ; 2012b 2012b ; Zhao et al., 2009b 2009b ; 2012a 2012a ; 2012b 2012b ; 2012c 2012c ). Mitochondrial DNA sequence data have been widely used in phylogenetic studies among animal taxa because of their matrilineal inheritance, lack of extensive recombination, and accelerated nucleotide substitution rates. Recently, we conducted mitochondrial 16S rDNA partial sequence analysis of five D. folliculorum isolates from the facial skin of Chinese individuals and two D. folliculorum isolates from Spain (one from facial skin and the other from eyelids) to identify intraspecies variation between the two geographic D. folliculorum isolates (Spain and China). The results indicated that there were no differences among the isolates from the two geographic areas or from the two tissue sources (Zhao and Wu, 2012b 2012b ). However, protein coding genes (such as cytochrome oxidase subunit 1 ( cox1 ), cytochrome oxidase subunit 2 ( cox 2), nicotinamide adenine dinucleotide phosphate dehydrogenase subunit I ( NDI ), and nicotinamide adenine dinucleotide phosphate dehydrogenase subunit S ( NDS )), which have more rapid evolution rates than mitochondrial 16S rDNA, are more useful for the phylogenetic analysis of closely related species, subspecies and different geographic populations. In this study, we analyzed sequences of the partial mitochondrial cox1 gene to characterize D. folliculorum isolates from China and to compare them with those of specimens from Spain, to investigate their taxonomic status. The cox1 partial sequences of one D. brevis isolate and one D. canis isolate were also included and analyzed to establish a reference frontier for intraspecies and interspecies variations in D. folliculorum , because of the lack of a standard for intraspecific identification of D. folliculorum from different populations using mitochondrial cox1 . Materials and methods 1 Demodex mite collection 2 Five D. folliculorum isolates and one D. brevis isolate (Xi’an, China) were sampled by the cellophane tape method from sufferers’ facial skin, and one D. canis isolate (Xi’an, China) was sampled by scraping from the infection focus of a Tibetan mastiff. The adult of each isolate was collected separately using home-made needles, after being identified microscopically (magnification 10×4) (Li, 2009 2009 ), and then preserved in Eppendorf tubes (EPs) containing 20 μl dilution buffer. The mitochondrial cox1 fragments of two D. folliculorum isolates (one from facial skin and the other from eyelids) from Spain were provided by Manuel DE ROJAS. DNA extraction 2 Genomic DNA of individual mites was extracted according to Zhao et al. ( 2012b 2012b ). DNA release additive (0.5 μl) was added to each tube and vortexed. After being incubated at room temperature for 5 min, the reagents were placed in 98 °C for 2 min, and finally preserved in EPs at −20 °C. Polymerase chain reaction (PCR) amplification of cox1 partial sequence 2 Specific primers (sense: 5′-GATTTTTTGGTCACCCAGAAG-3′; anti-sense: 5′-AGTGGAAGTGGGCTACGAC-3′) of the mitochondrial cox1 partial sequence were designed according to the correlated sequences (GenBank accession Nos. FN424247 and FN424248, Spain) and the primers were synthesized by Beijing AuGCT Biotechnology Co., Ltd., China. Extracted DNAs of D. canis , D. brevis , and D. folliculorum (Table 1 1 ) were amplified using an Applied Biosystems 2720 thermal cycler in 12.5 μl reaction volume containing 6 μl of 2× Premix Taq buffer, pH 8.3 (0.05 U/μl Taq DNA polymerase, 3 mmol/L MgCl 2 , 0.4 mmol/L each dNTP), 1 μl of 1 μmol/L of each primer, 1 μl of DNA template, and 3.5 μl of double distilled water. The amplification followed the thermal profile: pre-degeneration at 94 °C for 4 min; then 40 cycles of degeneration at 94 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 2 min; and finally extension at 72 °C for 10 min in a thermal cycler. The PCR products were separated by electrophoresis in 2% agarose gels (0.02 g/ml), prestained with ethidium bromide, at 120 V for 20 min in tris-borate-ethylenediaminetetraacetic acid (TBE) buffer, and visualized under UV light with 100 bp DNA ladder (TaKaRa) as the marker. Demodex mites were collected in this study Mite species Nation GenBank acc. No. Demodex folliculorum 1 * * China JF784002 Demodex folliculorum 2 * * China HQ844222 Demodex folliculorum 3 * * China JF784003 Demodex folliculorum 4 * * China JF784004 Demodex folliculorum 5 * * China JF784005 Demodex folliculorum 6 Spain FN424248 Demodex folliculorum 7 Spain (eyelids) FN424247 Demodex canis * * China JF731345 Demodex brevis * * China HQ844223 Cloning and sequencing of cox1 partial sequence 2 Expecting that the quantity of DNA extracted was too poor to be sequenced directly, cloning of PCR product was extended to obtain more DNA. cox1 fragments were purified using an OMEGA gel extraction kit, cloned after being linked with pMD18-T vector (TaKaRa), and then transformed into Escherichia coli (DH5α). Ampicillin screening and PCR after plasmid extraction were applied for the identification of clones containing cox1 fragments. At least two clones screened from six identical cox1 -positive clones per sample were sequenced by Beijing AuGCT Biotechnology Co., Ltd., China. The two sequences obtained were each aligned with the template used for primer design to confirm their consistency and reliability. If the sequencing failed or the sequence consistency was poor, the cloning and sequencing procedure was repeated. DNA sequence analysis 2 The nucleotide alignments of the cox1 sequences of the nine Demodex isolates obtained were conducted using ClustalX software version 1.8 (Thompson et al., 1997 1997 ) in the multiple alignment mode. The gene divergence of the pairwise cox1 partial sequences was estimated by DNAStar. Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (Tamura et al., 2007 2007 ) was employed to analyze the nucleotide composition, variable sites, parsim-info sites, and singleton sites, and to compute genetic distances based on the Maximum Composite Likelihood Model. Base composition bias and skew measures of the third-position nucleotides 2 MEGA 4.0 was further used to analyze transition/transversion and nucleotide composition of the third-position nucleotides. CG and AT content percentages, GC-skew (G−C)/(G+C) and AT-skew (A−T)/(A+T) (Perna and Kocher, 1995 1995 ) were computed and graphical representations of GC-skew and AT-skew were drawn in Excel 2003. Phylogenetic analysis 2 Demodex brevis (one isolate) and D. canis (one isolate) were taken as outgroups for the D. folliculorum isolates (five Chinese and two Spanish). A total of nine cox1 partial sequences from the Demodex were used to reconstruct the phylogenetic tree by maximum likelihood (ML) method in PhyML 3.0. Other methods (neighbour-joining (NJ), minimum evolution (ME), and maximum parsimony (MP)) were explored in MEGA 4.0 to confirm the reliability of phylogenetic relationships in the nine isolates of three Demodex species. Information on the cox1 sequences is listed in Table 1 1 . Results 1 PCR amplification and cloning 2 The mitochondrial cox1 fragments of the seven isolates of three Demodex species from Xi’an, China were amplified successfully, and shown by sequencing to be 429 bp long. We submitted the sequences to GenBank, and obtained the accession Nos. JF784002, HQ844222, JF784003, JF784004, JF784005, JF731345, and HQ844223. cox1 sequence analysis 2 The cox1 partial sequences (429 bp) of the seven Demodex isolates (five D. folliculorum , one D. brevis and one D. canis ) were aligned with those of the two D. folliculorum isolates from Spain (Fig. 1 1 ). DNAStar analysis showed a 97.4% (418/429) sequence identity and a 2.6% divergence in the five D. folliculorum isolates from the facial skin of Chinese individuals, and MEGA 4.0 analysis showed 418 identical sites and 11 variable sites (8 singleton and 3 parsim-info sites). The average contents of A, C, G, and T were 33.9%, 23.8%, 13.8%, and 28.5%, respectively, and the nucleotide frequencies were biased toward A+T, averaging 62.4%. The mean G+C contents of five D. folliculorum isolates from the facial skin of Chinese individuals and one from a Spanish individual were 37.6% and 36.8%, respectively, a 0.8% difference. The G+C contents of one D. folliculorum isolate from the eyelids of a Spanish individual, one D. canis isolate, and one D. brevis isolate were 37.3%, 38.0%, and 41.5%, respectively. The nucleotide data were grouped into triplets corresponding to the codons. D.f .: D. folliculorum ; D.c : D. canis ; D.b : D. brevis Divergence 2 Table 2 2 shows that in D. folliculorum species, the average pairwise sequence divergence was 1.24% among five isolates from Xi’an, China, 0.94% between one isolate from the facial skin of a Spanish individual and five from Chinese individuals, and 2.15% between one isolate from the eyelids and six isolates from the facial skin. The average pairwise sequence divergence of all seven D. folliculorum isolates was 1.43%. The interspecies divergence between the D. canis and D. folliculorum isolates was 14.53%, and between the D. brevis and the D. folliculorum isolates was 23.10%. Thus, interspecific divergence was significantly larger than intraspecies divergence in D. folliculorum . Above the diagonal: pairwise divergence; Below the diagonal: pairwise genetic distance Divergence=1−%identity. D.f .: D. folliculorum ; D.c : D. canis ; D.b : D. brevis Mite species D.f .1 D.f .2 D.f .3 D.f .4 D.f .5 D.f .6 D.f .7 D.c D.b D.f .1 1.9 1.4 1.2 0.9 1.4 2.9 14.4 23.3 D.f .2 0.019 2.1 0.7 0.9 0.9 2.4 15.0 23.7 D.f .3 0.014 0.022 1.4 1.7 1.7 2.1 14.4 23.0 D.f .4 0.012 0.007 0.014 0.2 0.2 1.7 14.4 23.0 D.f .5 0.009 0.009 0.017 0.002 0.5 1.9 14.7 23.3 D.f .6 0.014 0.009 0.017 0.002 0.005 1.9 14.4 23.0 D.f .7 0.029 0.024 0.022 0.017 0.019 0.019 14.4 22.4 D.c 0.167 0.175 0.167 0.167 0.171 0.167 0.167 26.0 D.b 0.309 0.313 0.304 0.304 0.308 0.304 0.293 0.354 Genetic distance 2 The pairwise genetic distances were computed by MEGA 4.0 (Table 2 2 ). In D. folliculorum , the average pairwise genetic distance of five isolates from the facial skin was 0.0125 among Chinese individuals, 0.0094 between one isolate from the facial skin of a Spanish individual and five isolates from Chinese individuals, and 0.0217 between one isolate from the eyelids and six isolates from the facial skin. The average intraspecific genetic distance among the seven isolates was 0.014. The genetic distance between isolates from different species was 10 times larger than that of isolates from the same species. Interspecific/intraspecific genetic distance was 12.05 (0.169/0.014) between D. folliculorum and D. canis , and 21.79 (0.305/0.014) between D. folliculorum and D. brevis . The third-position nucleotides analysis 2 Of the 142 third-position nucleotides (Fig. 1 1 ) in D. folliculorum species, the mean transition/transversion rate was 2.7 (3/1) among five isolates from the facial skin of Chinese individuals, 3.1 (3/1) between one isolate from the facial skin of a Spanish individual and five isolates from Chinese individuals, and 4.4 (3/1) between one isolate from the eyelids and six isolates from the facial skin. The transition/transversion rate between the D. folliculorum and D. canis isolates was 1.2 (8/7) and between the D. folliculorum and D. brevis isolates was 0.7 (7/9). The G+C contents of seven D. folliculorum isolates are shown in Table 3 3 . GC-skew and AT-skew data are presented in Fig. 2 2 . D.f .: D. folliculorum ; D.c : D. canis ; D.b : D. brevis Mites T (%) C (%) A (%) G (%) A+T (%) G+C (%) D.f .1 19.0 34.5 45.1 1.4 64.1 35.9 D.f .2 19.7 33.1 45.8 1.4 65.5 34.5 D.f .3 19.0 33.8 46.5 0.7 65.5 34.5 D.f .4 19.7 33.1 46.5 0.7 66.2 33.8 D.f .5 19.7 33.1 45.8 1.4 65.5 34.5 D.f .6 20.4 32.4 46.5 0.7 66.9 33.1 D.f .7 21.1 31.7 46.5 0.7 67.6 32.4 D.c 24.7 34.5 40.8 0.0 65.5 34.5 D.b 12.7 38.0 49.3 0.0 62.0 38.0 D.f .: D. folliculorum ; D.c : D. canis ; D.b : D. brevis Phylogenetic analysis 2 The phylogenetic trees were reconstructed for the nine cox1 partial sequences from isolates of three Demodex species. The topological structures of NJ, ME, and MP trees were nearly the same as that of the ML tree (Fig. 3 3 ). Two geographical D. folliculorum isolate groups, five isolates from the facial skin of Chinese individuals and one from a Spanish individual, were gathered first, then one isolate ( D. folliculorum 7) from the eyelids of a Spanish individual, and finally D. brevis and D. canis . The numbers on each node represent the bootstrap supports. D. folliculorum isolates from China (●); D. folliculorum isolates from Spain (★); D. canis (▲); D. brevis (♦) Discussion 1 A subspecies is a category ranking immediately below a species and designates a population of a particular geographic region or host. It is genetically distinguishable from other such populations of the same species and capable of interbreeding with them. A species will either be recognized as having no subspecies at all, or having two or more, but never only one. Based on the definition of a subspecies, we hold doubts about the validity of the facial D. folliculorum subspecies proposed by Xie et al. ( 1982 1982 ). First, D. folliculorum is distributed worldwide, and few reports of polymorphism in D. folliculorum resulting from geography or race of human hosts can be found. There are ample studies containing the term “ D. folliculorum ” in PubMed but none with “ D. folliculorum folliculorum ”, and no reports on D. folliculorum sinensis can be found in the China National Knowledge Infrastructure (CNKI), suggesting that the subspecies of D. folliculorum has not been acknowledged. Second, the validity of their results and conclusions was impaired because of the scarcity of hosts and samples. The mite samples in their study were collected from a limited number of hosts, which might not reflect the general morphological characteristics of the mites in China or serve as being representative of “ D. f. sinensis ”. Therefore, we suspect that the differences in morphological characteristics between “ D. f. folliculorum ” and “ D. f. sinensis ” proposed by Xie et al. ( 1982 1982 ) might reflect polymorphism in geographically isolated populations but not differences between subspecies. Whether subspecies, based on their morphological differences, exist in D. folliculorum remains to be confirmed. Here we provide the first comparison of cox1 partial sequences from nine isolates of three Demodex species. Subspecies differentiation of D. folliculorum from the facial skin has not been found at the molecular level, which is in accord with our deduction of polymorphism in geographical isolates. The extent of sequence divergence used to establish intraspecific differentiation in animals is rarely greater than 2% and mostly less than 1% (Avise and Walker, 1999 1999 ; Hebert et al., 2003 2003 ; Tsao and Yeh, 2008 2008 ). In D. folliculorum from the facial skin, the average pairwise divergence between an isolate from Spain and five isolates from China was 0.94%, smaller than that (1.20%) of five isolates from Xi’an China. The average pairwise genetic distance between one isolate from Spain and five isolates from China was 0.0094, which was not only smaller than that (0.0125) of five isolates from Xi’an, China, but also in accordance with the standard that interspecific genetic distance is rarely less than 0.020 (Liu et al., 2010 2010 ). Of the 142 third-position nucleotides, the mean transition/transversion rate among five isolates from China was 2.7 (3/1), and between one isolate from Spain and five isolates from China was 3.1 (3/1), both larger than the intraspecies standard of 2.0 (Kumar et al., 2008 2008 ; Zhou et al., 2010 2010 ). All the results indicate that no subspecies differentiation exists in D. folliculorum from the facial skin of these two geographical groups. However, a population difference was found between D. folliculorum isolates from facial skin and eyelids. The average pairwise divergence between one isolate from eyelids and six from facial skin was 2.15%, larger than the intraspecies standard of 2.0%. The average pairwise genetic distance between them was 0.0217, larger than the intraspecies standard of 0.020, both results indicating that a difference might exist between the two D. folliculorum populations from different facial tissues (eyelids and facial skin). The distinct local internal environment of D. folliculorum may be related to this differentiation. Our results are in accordance with the proposal of de Rojas et al. ( 2012a 2012a ) that cox1 gene sequences can help to identify different populations (in the facial skin and eyelids in this study), which were morphologically very close and difficult to separate by classical methods. However, as the same D. folliculorum sample from the eyelids of a Spanish individual was used, the general applicability of our results was impaired because of the scarcity of samples, and therefore needs to be confirmed. Further examination of cox1 partial sequences using ML phylogenetic tree analysis (Fig. 3 3 ) showed that three Demodex species gathered separately, suggesting that they are three independent species. In D. folliculorum species, six isolates from the facial skin gathered together and did not form sister clades. This result supported our conclusion that the D. folliculorum isolates from the facial skin were from the same population and no geographical differences existed. One isolate from the eyelids of a Spanish individual formed sister clades with six isolates from the facial skin, supporting our conclusion that differentiation may exist between populations of D. folliculorum from eyelids and facial skin. 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Mar Fish , 2010 . 32 ( 4 ): 351 - 355 . (in Chinese) . On phylogenetic relationships of Teraponidae in coastal waters of China Compliance with ethics guidelines Ya-e ZHAO, Jun-xian MA, Li HU, Li-ping WU, and Manuel DE ROJAS declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1200363 Project (No. 81271856) supported by the National Natural Science Foundation of China 2012-12-29 2013-08-04 2013-05-28 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2013 2013 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 09 14